Fluorescent labeling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer

ABSTRACT

The present invention provides low molecular weight fluorescent labeling complexes with large wavelength shifts between absorption of one dye in the complex and emission from another dye in the complex. These complexes can be used, for example, for multiparameter fluorescence cell analysis using a single excitation wavelength. The low molecular weight of the complex permits materials labeled with the complex to penetrate cell structures for use as probes. The labeling complexes are synthesized by covalently attaching through linkers at least one cyanine fluorochrome to another low molecular weight fluorochrome to form energy donor-acceptor complexes. Resonance energy transfer from an excited donor to fluorescent acceptor provides wavelength shifts up to 300 nm. The fluorescent labeling complexes preferably contain reactive groups for the labeling of functional groups on target compounds, such as derivatized oxy and deoxy polynucleic acids, antibodies, enzymes, proteins and other materials. The complexes may also contain functional groups permitting covalent reaction with materials containing reactive groups.

This invention was made with Government support under NIH-NS-19353 andNIH GM 34639.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to fluorescent labeling complexes, andmore particularly to low molecular weight fluorescent complexes withlarge Stokes shifts.

2. Description of the Invention Background

Fluorescence labeling is an important technology for detectingbiological molecules. For example, antibodies can be labeled withfluorescent dyes. The binding of antibodies to their specific targetmolecules can then be monitored on the basis of a fluorescence signal,which may be detected with a spectrometer, immunofluorescenceinstrument, flow cytometer, or fluorescence microscope. In a similarway, DNA sequences can be detected with fluorescence detectioninstruments after the DNA has been "hybridized" with a complementary DNAsequence that has been labeled with a fluorescent dye.

Very bright and water soluble fluorescent labeling reagents areimportant for sensitive detection of labeled antibodies, DNA probes,ligands, cytokins, drugs, lipids, metabolites and other molecules andcompounds of interest. Multiparameter analysis using fluorescent labelswith distinctly different emission wavelengths further increase theimportance of this technology by providing a powerful tool forcorrelating multiple antigenic or genetic parameters in individualcells. In epifluorescence microscopy, a continuous light source withdifferent sets of excitation and emission filters are used to excite anddetect each fluorescent species. This approach works especially well ifthe absorption and emission wavelengths of each of the fluorophores arerelatively close together (e.g. Stokes shifts of 15-30 nm). Most of thehighly fluorescent, low molecular weight fluorochromes like the cyaninesand xanthenes have narrow absorption and emission peaks and small Stokesshifts. Up to 5 separate fluorescent labels have been analyzed on thesame specimen by microscopy using epifluorescence filter sets asdescribed in DeBiasio, R., Bright, G. R., Ernst, L. A., Waggoner, A. S.,Taylor, D. L. "Five-parameter fluorescence imaging: Wound healing ofliving Swiss 3T3 cells," Journal of Cell Biology, vol. 105, pp.1613-1622(1987).

Flow cytometers and confocal microscopes are different from microscopesequipped with separate epifluorescence filter sets, in that they utilizelasers with defined wavelengths for fluorescence excitation. While it iseasy to find a single fluorophore that can be efficiently excited at aparticular laser wavelength, it is difficult to find additionalfluorescent labels with large enough Stokes shifts to provide emissionwell separated from that of the first fluorophore. The naturallyoccurring phycobiliproteins are a class of multichromophore fluorescentphotosystem proteins that have large wavelength shifts. See, Oi, V. T.,Glazer, A. N., Stryer, L. "Fluorescent phycobiliprotein conjugates foranalyses of cells and molecules," Journal of Cell Biology, vol.93,pp.981-986 (1982). These can be covalently coupled to antibodies andhave become widely used in flow cytometry for 2 color lymphocyte subsetanalysis. R-phycoerythrin (R-PE), a photosystem protein containing 34bilin fluorophores which can be excited at 488 nm with the widelyavailable argon ion laser, has been especially useful. It fluorescesmaximally at 575 nm. R-PE and fluorescein can both be excited at 488 nm,but R-PE can readily be discriminated with optical band-passinterference filter sets from the fluorescein signal, which appears at525 nm. Recently, 3-color immunofluorescence by flow cytometry hasbecome possible through the development of tandem conjugate labelingreagents that contain a reactive cyanine fluorescent dye which isexcited at 488 nm and fluoresces at 613 nm, and is sold commerciallyunder the name Cychrome. See, U.S. Pat. No. 4,876,190 and Waggoner, A.S., Ernst, L. A., Chen, C. H., Rechtenwald, D. J., "PE-CY 5; A newfluorescent antibody label for 3-color flow cytometry with a singlelaser," Ann. NY Acad. Sci., vol.677, pp.185-193 (1993). With thesetandem fluorophores, energy transfer from excited R-PE to the Texas Redor the reactive pentamethine cyanine known as CY5 leads to fluorescenceat 620 nm or 670 nm, respectively.

The phycobiliprotein-based labels are very fluorescent and provideexcellent signals in 2 and 3-parameter experiments for detection of cellsurface antigens. However, these reagents have not been widely utilizedfor measurement of cytoplasmic antigens or for detection of chromosomalmarkers by fluorescence in situ hybridization because their large size(MW=210,000 Daltons) limits penetration into dense cell structures.

There is a need for a new class of low molecular weight fluorescentlabels that will provide multicolor fluorescence detection using singlewavelength excitation. There is a further need for several suchflourescenct labels each of which can be excited optimally at aparticular laser wavelength but that fluoresce at significantlydifferent wavelengths.

SUMMARY OF THE INVENTION

The present invention provides a low molecular weight fluorescentlabeling complex which includes a first, or donor, fluorochrome havingfirst absorption and emission spectra, and a second, or acceptor,fluorochrome having second absorption and emission spectra. At least oneof the first or second fluorochromes is a cyanine dye. The wavelength ofthe emission maximum of the second fluorochrome is longer than thewavelength of the emission maximum of the first fluorochrome, and aportion of the absorption spectrum of the second fluorochrome overlaps aportion of the emission spectrum of the first fluorochrome for transferof energy absorbed by the first fluorochrome upon excitation with lightto the second fluorochrome.

The complex also includes a linker for covalently attaching thefluorochromes to permit resonance energy transfer between the first andthe second fluorochromes. The linker may be flexible and in a preferredembodiment, separates the fluorochromes by a distance that providesefficient energy transfer, preferably better than 75%. The linker may beabout 2 to 20 bond lengths. A preferred length for the linker is lessthan 70 Angstroms (7 nm), and more preferably, less than 20 Angstroms (2nm). In the case of flexible linkers, particularly when the labelingcomplexes are in solution, the relative orientations of the first andsecond fluorochromes changes as the linker flexes.

The first fluorochrome preferably has an extinction coefficient greaterthan 20,000 Liters/mole cm and preferably greater than 50,000Liters/mole cm and the second fluorochrome has a fluorescence quantumyield greater than or equal to about 0.05. Quantum yield is generallyrelated to a molecule's rigidity or planarity and indicates themolecules propensity to fluoresce, i.e. to give off energy as light,rather than as heat when energy is provided to the molecule. Thecombined molecular weight of the fluorochromes and the linker is in therange of 500 to 10,000 Daltons.

The complex includes a target bonding group capable of forming acovalent: bond with a target compound to enable the complex to label thetarget, such as a carrier material or a biological compound. The targetbonding group may be a reactive group for reacting with a functionalgroup on the target compound or molecule. Alternatively, the complex maycontain the functional group and the target may contain the reactiveconstituent. The reactive group is preferably selected from the groupconsisting of succinimidyl ester, isothiocyanates, dichlorotriazine,isocyanate, iodoacetamide, maleimide, sulfonyl halide, acid halides,alkylimidoester, arylimidoester, substituted hydrazines, substitutedhydroxylamines, carbodiimides, and phosphoramidite. The functional groupmay be selected from the group consisting of amino, sulfhydryl,carboxyl, hydroxyl and carbonyl. The target may be antibody, antigen,protein, enzyme, nucleotide derivatized to contain one of an amino,hydroxyl, sulfhydryl, carboxyl or carbonyl groups, and oxy or deoxypolynucleic acids derivatized to contain one of an amino, hydroxy,sulfhydryl, carboxyl or carbonyl groups, cells, polymer particles orglass beads. In the alternative embodiment, the target may bederivatized to contain the reactive groups identified above to formcovalent bonds with the functional groups on the complex.

The complex preferably also includes water solubilizing constituentsattached thereto for conferring a polar characteristic to the complex.They are preferably attached to the aromatic ring of the cyaninefluorochrome. The water solubilizing constituents must be unreactivewith the target bonding group of the complex. The solubilizingconstituents are preferably selected from the group consisting of amide,sulfonate, sulfate, phosphate, quaternary ammonium, hydroxyl andphosphonate. Water solubility is necessary when labeling protein and oxyor deoxy nucleic acids derivatized with amino groups or sulfhydrylgroups in aqueous solutions. A less polar form of the energy transfercompound may bind noncovalently to DNA by intercalation between basepairs or by interaction in the minor groove of DNA. Such compounds wouldbe useful for DNA quantification or localization.

In addition to the embodiment of the invention which includes a singledonor and a single acceptor fluorochrome, the fluorescent labelingcomplex may further include a third fluorochrome having third absorptionand emission spectrum and covalently attached to the secondfluorochrome. The wavelength of the emission maximum of the thirdfluorochrome is longer than the wavelength of the emission maximum ofthe second fluorochrome, and a portion of the emission spectrum of thesecond fluorochrome overlaps a portion of the absorption spectrum of thethird fluorochrome for transferring energy absorbed from the firstfluorochrome to the second fluorochrome to the third fluorochrome.Energy transfer procedes consecutively, i.e. in series, from the firstto the second to the third fluorochrome.

In an alternative embodiment, the complex may include a plurality of thefirst fluorochromes each covalently linked to the second fluorochromeand each capable, upon excitation with light, of transferring energy tothe second fluorochrome. In another embodiment, the complex may includea plurality of the second fluorochromes each covalently linked to thefirst fluorochrome and each capable of accepting energy from the firstfluorochrome when the first fluorochrome is excited by light. Theplurality of first or second fluorochromes may be the same molecule ormay be different. For example, there may be several donor fluorochromeswhich are excitable at different wavelengths to accommodate differentexcitation light sources. Energy transfer procedes in parallel in theseembodiments.

The labeling complexes of the invention are synthesized preferably bycovalently linking cyanine fluorochromes to other cyanine fluorochromesto form energy donor-acceptor complexes. Cyanine fluorochromes areparticularly useful for preparation of these complexes because of thewide range of spectral properties and structural variations available.See, for example, Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis,C., Waggoner, A. S. "Cyanine dye labeling reagents. Sulfoindocyaninesuccininmidyl ester," Bioconjugate Chemistry, vol.4, pp.105-111 (1993)and U.S. Pat. No. 5,268,486 to Waggoner et al., the disclosure of whichis incorporated herein by reference.

The invention also includes a reagent and a method for making thereagent including incubating the fluorescent water soluble labelingcomplex described above with a carrier material. One of the complex orthe carrier material has a functional group that will react with areactive group of the other of the complex or the carrier material toform a covalent bond there between. The carrier material can be selectedfrom the group consisting of polymer particles, glass beads, cells,antibodies, antigens, protein, enzymes, nucleotide derivatized tocontain one of an amino, sulfhydryl, carbonyl, carboxyl or hydroxylgroups, and oxy or deoxy polynucleic acids derivatized to contain one ofan amino, sulfhydryl, carboxyl, carbonyl or hydroxyl groups.Alternatively, the carrier material may contain the reactive groups andthe fluorescent labeling complex of the invention may contain any of theaforementioned functional groups that will react with the reactivegroups to form covalent bonds.

In an alternative embodiment, the fluorescent complexes of the inventionneed not have a reactive group when used to noncovalently bind toanother compound. For example, the complex may be dissolved, then mixedin an organic solvent with a polymer particle, such as polystyrene, thenstirred by emulsion polymerization. The solvent is evaporated and thefluorescent dye complex is absorbed into the polystyrene particles.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood by reference to thedrawings in which:

FIG. 1 is a schematic illustration of the overlapping absorption andemission spectra of four cyanine fluorochromes that can be used in theenergy transfer labeling complex of the present invention;

FIG. 2(a) is the absorbance spectrum for the complex consisting of atrimethine and a pentamethine cyanine in a 1:1 ratio in methanol andFIG. 2(b) is the absorbance spectrum for the complex consisting of atrimethine and a pentamethine cyanine in a 2:1 ratio in methanol;

FIG. 3 illustrates the absorption spectra of two fluorescent labelingcomplexes, complex 1 (solid) in methanol, comprised of one cyanine donorand one cyanine acceptor, and complex 6 (dotted) in methanol, comprisedof two cyanine donors and one cyanine acceptor;

FIGS. 4(a) and (b) illustrate the absorbance (solid) and fluorescence(dotted) spectra of complex 1 of the invention made of trimethine andpentamethine cyanine dyes in (a) methanol and (b) PBS;

FIG. 5 illustrates a normalized excited spectra of the complex 1 in PBS(solid line), methanol ( ), glycerol ( ), and complex 1-streptavidinconjugate in PBS ( - - - );

FIG. 6 illustrates the absorbance spectra in PBS of Sheep IgG-complex 1conjugates at various dye:protein ratios demonstrating that no dimerformation involving either donor or acceptor is evident with increasingdye:protein ratios; and,

FIG. 7 illustrates the two color flow cytometry analysis of humanlymphocytes labeled with anti-CD4-PE and anti-CD3-streptavidin- complex1 to mark the helper cell subset of T-cells and total T-cell subset,respectively, showing a subset of complex 1 labeled cells without the PEsignal and a second subset of complex 1 labeled cells that is PEstained;

FIG. 8 is the absorbance spectrum for the complex consisting offluorescein and a trimethine cyanine;

FIG. 9 is the fluorescence spectrum for the complex of FIG. 8;

FIG. 10 is the absorbance spectrum for the complex consisting offluorescein, a trimethine cyanine and a pentamethine cyanine;

FIG. 11 is the fluorescence spectrum for the complex of FIG. 10;

FIG. 12 is the absorbance spectrum for the complex consisting offluorescein, a trimethine cyanine and a heptamethine cyanine; and,

FIG. 13 is the fluorescence spectrum for the complex of FIG. 12.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides low molecular weight, preferably watersoluble, fluorescent labeling complexes with large excitation-emissionwavelength shifts or Stokes shifts. For purposes of the presentspecification, the Stokes shift of the fluorescent complex is theabsolute difference in nanometers between the absorbance maximum of thelowest light absorber of the complex and the fluoresence of the longestwavelength emitter of the complex. The complexes, as briefly explainedabove, contain two or more fluorochromes linked together for transfer ofenergy from a shorter wavelength to a longer wavelength. As shownschematically in FIG. 1, the first, donor fluorochrome absorbs energyupon excitation at an excitation wavelength (solid line) within itsabsorbance spectrum and emits energy at a wavelength within its emissionspectrum (broken line). When linked at an appropriate orientation to asecond, acceptor fluorochrome, the donor fluorochrome transfers, ordonates, the energy of its excited state to the acceptor fluorochrome ata wavelength within the absorbance spectrum (solid line) of the acceptorfluorochrome. The acceptor fluorochrome absorbs the donated energy andemits it at a wavelength within its emission spectrum (broken line),which as shown, is longer than the longest wavelength of the emissionspectra of the donor fluorochrome. It is important, therefore, that theemission spectrum of the donor fluorochrome overlap with the absorptionspectrum of the acceptor fluorochrome. The overlapping areas are shownby hatched lines. The greater the overlap, the more efficient the energytransfer.

The complexes include at least one cyanine fluorochrome, and preferablyat least one polymethine cyanine dye. The cyanines are particularlyuseful due to the wide range of spectral properties and structuralvariations available. Several such complexes will be described forpurposes of this detailed description. Other low molecular weightfluorochromes in addition to the cyanine fluorochromes, such as thefluoresceins, pyrene trisulfonates, which are sold under the trade markcascade blue, rhodamines and derivatives of the bispyrrometheneboron-difluoride dyes, such as 3,3',5,5'-tetramethyl2,2'-pyromethene-1,1'-boron-difluoride, sold under the trademark BODIPYby Molecular Probes, Inc., can be used to form the fluorescent labelingcomplexes of the invention. BODIPY analogs are disclosed in U.S. Pat.No. 4,774,339, 5,187,223, 5,248,782 and 5,274,113, all to Haugland andKang, as well as in "Handbook of Fluorescent Probes and ResearchChemicals" compiled by Haugland and published by Molecular Probes, Inc.

The fluorescent labeling complexes of the invention have low molecularweights and can be readily conjugated to antibodies, other proteins, andDNA probes. Low molecular weight as used herein shall mean that thecombined molecular weight of the fluorochomes and linker of the complexis between about 500 and 10,000 Daltons, and for a two fluorochromecomplex, preferably in the range of 1000 to 2500 Daltons. Therefore,these labeled species will have much greater penetration intointracellular environments than is possible with the largephycobiliprotein labels currently in use. The low molecular weightfluorescent labeling complexes of the invention should be valuable notonly for flow cytometry, but also for laser confocal microscopy and forother detection systems requiring multicolor detection with singlewavelength excitation.

Many structural varieties and modifications of cyanines are possible. Byvarying the number of carbons in the methine bridge of the cyanine dyesand the heteroatoms or other constituents on the cyanine dyes, a varietyof different spectral qualities can be achieved. The cyanine dyes areespecially well adapted for the analysis of a mixture of componentswherein dyes of a variety of emission wavelengths are required becausespecific cyanine and related dyes can be synthesized having a wide rangeof excitation and emission wavelengths. Specific cyanine and relateddyes have specific excitation and emission wavelengths can besynthesized by varying the number of methine groups or by modifying thecyanine ring structures. In this manner, it is possible to synthesizedyes having particular excitation wavelengths to correspond to aparticular excitation light source, such as a laser, e.g., a HeNe laseror a diode laser. Therefore, energy transfer labels can be made thatabsorb and emit efficiently at most wavelengths in the visible region ofthe spectrum. Commonly used sources of excitation excite at laser line488 nm. Therefore, that exemplary excitation wavelength will be used forpurposes of the description of the invention. Those in the art willrecognize that other energy transfer labels can be made for specificexcitation sources without departing from the scope of this invention.

The energy transfer between donor and acceptor fluorochromes that areappropriately selected and linked can be very efficient. The complexesprepared and described herein show energy transfer ranging from 50 to99% efficiency. Energy transfer efficiency depends on several factorssuch as spectral overlap, spatial separation between donor and acceptor,relative orientation of donor and acceptor molecules, quantum yield ofthe donor and excited state lifetime of the donor.

Complexes may be constructed using rigid linkers that optimally orientthe transition moments of the donor and acceptor chromophores.Alternatively, the linker may be flexible. For optimal energy transferto occur, the transition moments of the first and second fluorochromesare oriented relative to each other in a nonperpendicular direction.Transition moments positioned generally parallel or in tandem relativeto each other provide efficient transfer. In practice, the fluorochromesare not in a static position. The nonrigid linker covalently binding thefluorochromes flexes, particularly when the complexes are in solution.The transition moments of the fluorochromes will change as the linkerflexes, but, provided the donor and acceptor transition moments arenonperpendicular during the excited state lifetime of the donor, energytransfer will occur.

Shorter linkers would enhance transfer, since efficiency varies as theinverse 6th power of separation of the centers of the chromophoresaccording to Forster's Equation:

    ET∝K.sup.2 Φ.sub.D J/R.sup.6 τ.sub.D

where ET is energy transfer; K² is the relative orientation of donor andacceptor transition moments; Φ_(D) is the quantum yield of the donormolecule; R is the distance between the centers of the donor andacceptor fluorochromes; J is the overlap between the emission spectrumof the donor and the absorption spectrum of the acceptor fluorochromes;and τ_(D) is the excitated state life time of the donor molecule. See,Forster, T., "Intermolecular Energy Transfer and Fluorescence," Ann.Physik., vol.2, p.55 (1948). The distance R between the centers of thetwo fluorochromes, e.g., in a complex having two cyanine dyes, themiddle of the methine bridge of one cyanine to the middle of the methinebridge of the second cyanine, along the length of the compounds may befrom 10 to about 80 Angstroms. The length of the linker connecting thefluorochromes, as used herein, is different from the distance R. Thelinker should permit resonance energy transfer between thefluorochromes. The fluorochromes should not interact chemically or formsecondary bonds with each other. A preferred length for the linker isless than 70 Angstroms (7 nm), and more preferably, less than 20Angstroms (2 nm). In terms of bond length, the linker may be from 2 to20 bond lengths. For example, if the linker includes an alkyl chain,--(CH₂)_(n) --, the carbon number n may be from 1 to about 15. As nexceeds 15, the efficiency of the energy transfer decreases. The linkermay include part of the constituents extending from the cyanine dye. Inother words, the linker is attached to the dye chromophore, but is not apart of it. For example, referring to the linker shown in Table 1, someextend from the ring N in one cyanine to a functional group on thebenzene ring of the other cyanine. Some extend between functional groupson the benzene rings of linked dyes. The linker is placed on one cyaninedye before the dye linker combination is attached to the second dye.With a relatively short linker and optimal orientation, there may beefficient resonance energy transfer even when the spectral overlapbecomes small. Therefore, it is possible to obtain large wavelengthshifts even when only two chromophores are used in the complex.

The fluorescent labeling complexes include groups capable of formingcovalent bonds with corresponding groups on target compounds.Preferably, reactive groups are on the complex and functional groups areon the target compound or molecule. However, those skilled in the artwill recognize that the functional groups may be placed on the complexand the reactive groups may be on the target.

The reactive groups of the complexes of the invention includesuccinimidyl esters, isothiocyanates, dichlorotriazine, isocyanate,iodoacetamide, maleimide, sulfonyl halide, alkylimidoester,arylimidoester, carbodiimide, substituted hydrazines, hydroxylamines,acid halides and phosphoramidite. The reactive groups will form covalentbonds with one or more of the following functional groups: amine,hydroxyl, sulfhydryl, carboxyl and carbonyl.

To promote water solubility, water solubilizing constituents may beattached to the complex or to the linker. They include amide, sulfonate,sulfate, phosphate, quaternary ammonium, hydroxyl and phosphonategroups. Sulfonate or sulfonic acid groups attached directly to thearomatic ring of the cyanine fluorochrome are preferred.

Examples of some of the dyes that can be used as donor and acceptorfluorochromes in the fluorescent labeling complexes of the invention areshown in Table 1 below:

                                      TABLE 1                                     __________________________________________________________________________                                                 CASCADE BLUE                        -                                                                                                                       FITC ##                             -                                                                                                                       CY3NH.sub.2                         -                                                                                                                       CY3(NH.sub.2).sub.2                 -                                                                                                                       CY3NH.sub.2 SO.sub.3                                                          -                                                                             CY3--O(SO.sub.3).sub.2                                                        -                                                                             CY5(SO.sub.3).sub.2                 -                                                                                                                       CY5(COOH)                           -                                                                                                                       CY7(SO.sub.3).sub.2              __________________________________________________________________________

Additional cyanines for use in the complexes of the invention are therigidized monomethine cyanines disclosed in the copending applicationsof the Ratnakar Majumdar, Bhalchandra Karandikar and Alan S. Waggonerentitled "Rigidized Monomethine Cyanines" Ser. No. 08/474,056 filed Jun.7, 1995, and "Monomethine Cyanine Rigidized by a Two-Carbon Chain", Ser.No. 08/474,057 filed Jun. 7, 1995, the disclosures of which areincorporated herein by reference. The monomethine rigidized dyes havethe general structures ##STR10## The boron rigidized monomethine cyaninedyes have sharp distinct absorptive and emissive signals, and arephotostable. Certain of the boron-rigidized monomethine cyaninesmaximally absorb and emit light at wavelengths between 400-500 nm orless and fluoresce in the blue region of the visible spectrum.

Experiments have demonstrated that for obtaining exceptionally largeexcitation-emission wavelength shifts, it is possible to use sequentialenergy transfer steps in the complex. For example, three chromophoreshave been linked to provide maximal emission at the wavelength of acyanine dye, the heptamethine cyanine designated CY7 in Table 1 above(780 nm) with excitation at 488 nm. The initial donor was fluoresceinand the intermediate fluorophore in this complex was a trimethinecyanine dye designated generally as CY3. The fluorescein was excited at488 nm and transferred nearly 100% of its excited state energy to thetrimethine cyanine, which in turn transferred about 90% of its excitedstate energy to the CY7, fluorescing at 782 nm. The same efficiency wasobserved when a penthamethine cyanine, CY5 was used in place of CY7,with fluorescence at 667 nm. The development of such multichromophorecomplexes is particularly useful for multicolor detection systems.

Although several of the complexes show efficient energy transfer, theoverall quantum yield of these labeling complexes can be furtherimproved. For example, the use of acceptor dyes with quantum yieldshigher than that of CY5 (see Table 1) would improve the overall"brightness" of the complex.

An experiment was done to determine if two cyanine fluorochromes couldbe covalently linked for energy transfer. The cyanines used were CY5 andCY7 without reactive groups. The results demonstrated that the cyaninescould be covalently linked. The procedure is presented schematicallybelow. The dyes are represented by boxes.

EXAMPLE 1 ##STR11##

1. 5 mg cyanuric chloride (trichlorotriazine), 2 mg NaHCO₃, and 0.25 mLpurified dimethyl formamide (DMF) as solvent were mixed at 0° C. To thissolution was added 5 mg of amino-cyanine-5 dye represented above by thebox containing number 5, and the mixture was stirred at 0° C. for 10min. Stirring was continued overnight at room temperature. Thin layerchromatography (TLC) revealed one major spot and two minor spots; thelatter spots were determined to be impurities. UV-visible absorptionshowed a peak at 664 nm with a shoulder at 605 nm.

2. The reaction mixture was worked up by precipitation with ether. Adark blue powder was obtained. 0.3 mL DMF was added to dissolve thepowder. Then 2 mg of sodium bicarbonate and 4.7 mg of amino-CY7 dye,represented above by the box containg number 7, were added. The mixturewas stirred at room temperature for 24 hrs. Absorption peaks showed at650 nm (with a shoulder at 607 nm) and 761 nm. The reaction wasprecipitated by several washes with ether, providing a dark powder.

Following the initial success of the above experiments, six energydonor-acceptor complexes were prepared from cyanine fluorochromes inorder to investigate the energy transfer efficiency of such compounds.The structures of these complexes are shown in Table 2 and theirspectral properties are described in Table 3.

                                      TABLE 2                                     __________________________________________________________________________      #STR12##                                                                       -                                                                            #STR13##                                                                       -                                                                            #STR14##                                                                       -                                                                            #STR15##                                                                       -                                                                            #STR16##                                                                       -                                                                           ##STR17##                                                                    __________________________________________________________________________     "A" designates the fluorochrome that acts as the energy acceptor and "D"      designates the fluorochrome that acts as the energy donor.               

The energy transfer complexes shown in Table 2 are as follows: Complex1, CY3NH₂ SO₃ (Donor)+CY5(SO₃)₂ (Acceptor); Complex 2, CY3-O(SO₃)₂(Donor)+CY3NH₂ (Acceptor); Complex 3, CY3NH₂ (Donor)+CY5COOH (Acceptor);Complex 4, CY3NH₂ (Donor)+CY5(SO₃)₂ (Acceptor); Complex 5, CY3(NH₂)₂(Donor)+CY7(SO₃)₂ (Acceptor); Complex 6, 2 CY3NH₂ SO₃ (Donor)+CY5(SO₃)₂(Acceptor).

                  TABLE 3                                                         ______________________________________                                        Spectral Properties Of Cyanine Dyes Used As Precursors For The                  Fluorescent Energy Transfer Labeling Complexes Of The Invention.                                  Absorption                                                                             Emission                                                                              Quantum                                    maximum maximum yield                                                       Dye Solvent (nm) (nm) (Φ)                                               ______________________________________                                        Amine containing Cyanine Dyes                                                   CY3NH.sub.2                                                                             Methanol  552      569     0.05                                      PBS 548 563 0.05                                                             CY3(NH.sub.2).sub.2 Methanol 552 569 0.05                                      PBS 548 563 0.05                                                             CY3NH.sub.2 SO.sub.3 Methanol 556 573 0.08                                     PBS 548 653 0.09                                                           Carboxyalkyl containing Cyanine Dyes                                            CY5COOH   Methanol  658      685     0.22                                      PBS 648 667 0.13                                                             CY5(SO.sub.3).sub.2 Methanol 658 677 0.4                                       PBS 650 667 0.27                                                             CY3O(SO.sub.3).sub.2 Methanol 492 506 0.20                                     PBS 486 500 0.09                                                             CY7(SO.sub.3).sub.2 Methanol 758 789 ND.sup.a                                  PBS 750 777 ND.sup.a                                                       ______________________________________                                         .sup.a N.D. means not determined.                                             PBS means phosphatebuffered saline.                                      

The efficiency of energy transfer was estimated by calculating theamount of quenching of donor fluorescence that occurs (DQE) when theacceptor is attached. It is possible that some quenching could occur bypathways other than resonance energy transfer when the acceptor isbound. However, the cyanine donor preferred for the fluorescent labelingcomplexes of the present invention are relatively insensitive to theirmolecular environment. Furthermore, addition of large substituents totrimethine cyanines usually increase, rather than decreases, theirfluorescence. Therefore, DQE may be equal to the efficiency of energytransfer. The estimated energy transfer efficiencies based on DQEmeasurements ranged from 50% to 99% and the wavelength shifts betweenthe donor absorption maxima and the terminal acceptor emission maxima(DI) varied between 83 nm and 294 nm.

Two of the complexes, 1 and 6, are capable of absorbing light at theargon laser wavelength, 488 nm. Complex 1 contains a single donor andsingle acceptor, and complex 6 contains 2 donors per acceptor. Complex 1has 3 carboxyl groups and complex 6 has 4 carboxyl groups. These areconverted to succinimidyl active esters upon activation. FIG. 3 showsthe absorption spectra of complex 1 and complex 6 in methanol. Thespectra of the complexes are almost superimposable on absorption spectraobtaining by mixing 1:1 and 2:1 parts of the individual fluorochromes,CY3 and CY5, respectively, as shown in FIGS. 2(a) and 2(b).

Complex 1 was selected for further studies. As shown in FIGS. 4a and b,the absorbance (solid line) of complex 1 varies slightly in phosphatebuffer saline (FIG. 4b) and methanol (FIG. 4a) but fluorescence remainsunchanged. The emission of the donor component at 572 nm is very weakcompared with the emission of the acceptor at 675 nm, as would beexpected when energy transfer is efficient.

FIG. 6 demonstrates that sheep antibodies can be readily labeled withthe activated complex 1. Conjugates made of complex 1 conjugated tosheep IgG at various dye:protein ratios were tested. The lowestdye:protein ratio is represented by the line having its first peak (atabout 270 nm) at 0.8 and the highest dye:protein ratio is represented bythe line having its first peak (at about 270 nm) at a little less than0.4. No dimer formation involving either the donor or the acceptorfluorochromes was observed with increasing dye:protein ratios. Eachcomplex 1 contains up to 3 reactive groups. More reactive groups may beused provided no cross-liking occurs. It is important to use labelingconditions that avoid protein cross-linking which quench thefluorescence. Cross-linking by doubly activated cyanines has beenobserved previously by Southwick, P. L. et al., "Cyanine Dye LabelingReagents: Carboxymethylindocyanine succinimidyl esters," cytometry,vol.11, pp.418-430 (1990) and can be minimized by limiting theconcentration of protein to be labeled to approximately 1 mg/mL.

Upon binding to antibodies, the quantum yield of the complex wasenhanced three fold as shown in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    Spectral Properties Of Energy Transfer Complexes.                                    Absmax   Excitation                                                                              Quantum                                                                            Energy                                                                             Wavelength                                   nm Wavelength Emmax Yield transferred Shift                                  Dye (ex 10.sup.4) (nm) (nm) (Φ) (%) (nm)                                __________________________________________________________________________    Complex 1.sup.a                                                                      556(9.5), 652(14.3)                                                                    488   675 0.32 91   119                                           514 676 0.37 92 120                                                           600 673 0.49 -- --                                                          Complex 1.sup.b 536(16), 658 (16) 488 675 0.03 89 139                           514 673 0.04 89 137                                                           600 668 0.21 -- --                                                          Complex 558, 658 488 674 0.11 95 116                                          1PB5.c  514 673 0.13 95 116                                                     600 676 0.14 -- --                                                          Complex 1.sup.d 562, 658-- 488 674 0.19                                         514 674 0.32                                                                  600 674 0.39                                                                Complex 2.sup.a 490(13), 554 (9.5) 466 571 0.15 89  81                        Complex 3.sup.a 545(9.5), 658 (14.3) 514 679 0.08 83 133                      Complex 4.sup.a 550(9.4), 656 (14.2) 514 674 0.2  96 124                      Complex 5.sup.a 445(9.5), 754 (14.4) 520 782 N.D. 99 226                      Complex 6.sup.a 556(9.5), 652 (14.4) 488 674 0.23 49 118                        514 674 0.24 50 118                                                           600 674 0.34 -- --                                                          Complex 6.sup.b 548(20.0), 488 566 0.05 43 118                                 652(15.0) 514 564 0.05 38 116                                                  600 668 0.23 -- --                                                        __________________________________________________________________________     .sup.a In methanol,                                                           .sup.b In PBS                                                                 .sup.c Complex 1 on streptavidin, d/p = 4                                     .sup.d In glycerine                                                           N.D. means not determined                                                

It is believed that this occurs because the radiationless deactivationpathway of both the CY3 and CY5 components of complex 1 are reducedbecause of their restricted mobility when bound to the surface of theprotein. Other means of restricting conformational nobility are known toincrease the fluorescence efficiency of cyanine fluorochromes, asdescribed in Mujumdar, R. B. et al. "Cyanine dye labeling reagents.Sulfoindocyanine succininmidyl ester," Bioconjugate Chemistry, vol.4,pp.105-111 (1993). In fact, when complex 1 was dissolved in glycerine,the quantum yield increased by several fold as shown in Table 3.

Activated complex 1 can be used as a fluorescence label for 2 color flowcytometry experiments with 488 nm excitation. The scatter plot is shownin FIG. 7. Human T-lymphocytes were used to compare the complex 1 labelwith another 2-color reagent, R-Phycoerythrin, which also excites at 488nm and emits at 575 nm. Complex 1 labeled streptavidin(fluorochrome/protein ˜4) was used to detect biotinylated CD3 antibody,which marks all T-cells. In the same lymphocyte sample,Phycoerythrin(PE)-labeled anti-CD4 was used to mark the Helper-Cellsubset of the T-Cells. Thus, in the total lymphocyte population there isa population of cells that contain neither CD3 nor CD4 (i.e., CD3 andCD4 negative, shown in the lower left population of the 2-dimensionalscatter plot in FIG. 7), a subset of complex 1-labeled CD3-positivecells that do not have a Phycoerythrin signal (i.e., CD3 positive andCD4 negative, shown in the upper left population of FIG. 7), and a thirdsubset consisting of complex 1 labeled cells that are Phycoerythrinstained (i.e., CD3 and CD4 positive, shown in the upper right populationof FIG. 7). It is clear that complex 1 gave base-line separation of thepositive and negative cell populations, and that there was no spill overof complex 1 fluorescence into the Phycoerythrin channel. The complex 1fluorochrome gave a three time brighter signal when the fluorochrome wasexcited at 514 nm.

The method of synthesizing complex 1 is described in the example below.

EXAMPLE 2

Purification of Dyes: Purification of the fluorochromes was performed ona Spectra-Physica model SP8700 analytical HPLC unit equipped with aC8-RP column. Purification could also be achieved by conventional orflash column chromatography on commercially available C18-RP powder.Water-methanol mixtures were used for elution in all experiments. Dyeswere recovered from the fractions with a rotary evaporator at 60-70° C.without appreciable loss. The fluorochrome was passed with unknowncounterion composition through a Dowex-50W hydrogen, strongly acidiccation exchange column that had been previously washed with 0.1 Nsulfuric acid and then distilled water for further purification.

Spectroscopic Measurements and Analytical Determinations:Ultraviolet-visible spectra were measured with a Hewlett-Packard HP 8452diode array spectrophotometer. The proton NMR spectra were obtained withan IBM 300 FT-NMR spectrometer using D₂ O or DMSO d₆ as solvent.Fluorescence measurements were performed by using a SPEX Fluorolog 2system. Quantum yields were determined by well known techniques aspreviously described in Mujumdar, R. B. et al., "Cyanine Dye LabelingReagents Containing Isothiocyanate Groups," Cytometry, vol.10, pp.11-19(1989). NMR signals are described in δ by use of s for singlet, d fordoublet, t for triplet, q for quartet, and m for multiplet.

Cell Preparation and flow cytometry: Mononuclear leukocytes wereobtained by Histopaque density 1.077 separation of peripheral blood fromhealthy volunteers. The lymphocyte population was selected by flowcytometry based on forward and side scatter characteristics.Subpopulations were identified using specific monoclonal antibodies(CD4, staining T-helper cells and CD3, pan T-cell population). Optimalconcentration of complex 1 tagged antibody was determined by analyzingthe results of a dilution series. Direct immunofluorescence wasaccomplished by incubating the recommended amount of labeled antibodywith 1-2×10⁶ cells for 45 minutes at 4° C. Samples were then washedtwice in Hank's balanced salt solution (HBSS) containing 2% fetal bovineserum and 0.1% sodium azide. After the final wash, the cells wereresuspended in 1 mL of HBSS containing 1% paraformaldehyde and analyzedwithin one week. Flow cytometry measurements were made with a BectonDickinson FACS 440 dual laser flow cytometer equipped with a Consort 40data analysis system. The argon ion laser provided 400 mW of excitationat 488 nm. Fluorescence signals from complex 1 and R-Phycoerythrin werecollected using 670/13.5 nm and 575/26 nm band pass filters,respectively.

Calculation of donor quenching efficiency (DOE): Absorption andfluorescence spectra of the donor (alone) and the fluorescent labelingcomplex were obtained in order to determine the relative concentrationsof each in fluorescence experiments. Donor excitation was used to obtainemission spectra of both compounds. DQE was then calculated using

    DQE %=(1-F.sup.ET A/F A.sup.ET)×100%

where F is the fluorescence intensity of the donor alone, F^(ET) is theintensity of the donor of the complex, A is the absorbance at thewavelength of excitation (488 nm) of the donor alone and A^(ET) is theabsorbance at the wavelength of excitation (488 nm) of the fluorescentlabeling complex.

Syntheses of fluorochromes: Amino-cyanines (CY3NH₂, CY3(NH₂)₂ & CY3NH₂SO₃) and carboxyalkyl cyanines (CY5COOH, CY3O(SO₃)₂, CY5(SO₃)₂ &CY7(SO₃)₂) required as precursors for energy transfer fluorochromes weresynthesized by the methods previously described in Ernst, L. A. et al.,"Cyanine Dye Labeling Reagents For Sulfhydryl Groups", Cytometry,vol.10, pp.3-10 (1989), Hammer, F. M., THE CYANINE DYES AND RELATEDCOMPOUNDS, (Wiley pub. New York, 1964), Mujumdar, R. B. et al., "CyanineDye Labeling Reagents Containing Isothiocyanate Groups," Cytometry,vol.10, pp.11-19 (1989), Mujumdar, R. B. et al. "Cyanine dye labelingreagents. Sulfoindocyanine succininmidyl ester," Bioconjugate Chemistry,vol.4, pp.105-111 (1993), and Southwick, P. L. et al., "Cyanine DyeLabeling Reagents: Carboxymethy lindocyanine succinimidyl esters,"Cytometry, vol.11, pp.418-430 (1990). The synthesis and properties ofone amino-cyanine fluorochrome, CY3NH₂ SO₃, and its conjugation with thesuccinimidyl ester of CY5(SO₃)₂ to form complex 1 is described below.The spectral properties for all the fluorochromes are shown in Tables 3and 4 above. The unsymmetrical trimethinecarbocyanine, CY3-NH₂ SO₃, wassynthesized in four steps. Refer to Table 5 below for the structures(I)-(VI).

                  TABLE 5                                                         ______________________________________                                                                       X.sup.-                                                                         #STR18##                                        -                                                                                  R.sub.1   R.sub.2       X                                             ______________________________________                                        I         H           H             Br.sup.-                                    II CH.sub.2 Phth H Br.sup.-                                                   III CH.sub.2 Phth (CH.sub.2).sub.5 COOH Br.sup.-                              IV SO.sub.3.sup.- (CH.sub.2).sub.5 COOH --                                    #STR19##                                                                       - V, R.sub.1 = SO.sub.3.sup.-, R.sub.2 = CH.sub.2 Phth                       VI, R.sub.1 = SO.sub.3.sup.-, R.sub.2 = CH.sub.2 NH.sub.2 (CY3NH.sub.2           SO.sub.3)                                                                ______________________________________                                         ##STR20##                                                                     -                                                                        

1. Synthesis of5-Phthalimidomethyl-1-(ε-carboxypentynyl)-2,3,3-trimethylindole, (III).5-Phthalimidomethyl-2,3,3-trimethylindolenine (II) was synthesizedaccording to the procedure of Gale and Wilshire, "The amidomethylationand bromination of Fisher's base. The preparation of some newpolymethine dyes," Aust. J. Chem., vol.30, pp.689-694 (1977). PowderedN-hydroxymethylphthalimide (70 g, 0.4 mol) was added in small portionover a period of 45 min. to a stirred solution of2,3,3-trimethyl-(3H)-indolenine (I), (70 g, 0.44 mol) in concentratedsulfuric acid (360 mL) at room temperature. The solution was stirred for70 h at room temperature before being poured onto ice-water.Basification of the solution with conc. ammonium hydroxide gave a yellowpowder which was filtered and dried. (111 g, yield 80%, m.p. 180-182°C.). ¹ H NMR (DMSO d₆), δ, 7.8-7.95 (m, 4H, phthalimido), 7.4 (s, 1 H,4-H), 7.38 (d, 1 H, J=9.0 Hz, 6-H), 7.2 (d, 1 H, J=9 Hz, 7-H), 4.7 (s, 2H, --CH₂), 2.2 (s, 3 H, CH3), 1.2 (s, 6 H --(CH₃)₂).

This dry powder (10 g, 0.3 mol) and 6-bromohexanoic acid (9.1 g, 0.05mol) were mixed in 1,2-dichlorobenzene (25 mL) and heated at 125° C.)for 12 h under nitrogen. The mixture was cooled, 1,2-dichlorobenzene wasdecanted and the solid mass was triturated with isopropanol until freepowder was obtained. (11 g, yield 80%, m.p. 124-126° C.). ¹ H NMR (DMSOd6), δ, 7.8-7.95 (m, 4 H, phthalimido), 7.4 (s, 1 H, 4-H), 7.38 (d, 1 H,J=9.0 Hz, 6-H), 7.2 (d, 1 H, J=9 Hz, 7-H), 4.7 (s, 2 H, --CH₂), 4.5 (t,2 H, J=7.5 Hz, α--CH₂), 2.3 (t, 2 H, J=7 Hz, ε--CH₂), 1.99 (m, 2 H,β--CH₂), 2.3-1.7 (m, 4 H, γ--CH₂ and δ--CH₂ merged with s of 6H--(CH₃)₂).

2. Synthesis of1-(ε-Carboxypentynyl)-2,3,3-trimethylindoleninium-5-sulfonate (IV).Compound (IV) was synthesized according to the procedure describedpreviously by Mujumdar, R. B. et al., Bioconjugate Chemistry, (1993),supra. The potassium salt of 2,3,3-trimethylindoleninium-t-sulfonate (11g, 0.04 mol) and 6-bromohexanoic acid (9.8 g, 0.05 mol) were mixed in1,2 dichlorobenzene (100 mL) and heated at 110° C. for 12 h undernitrogen. The mixture was cooled. 1,2-dichlorobenzene was decanted andthe solid mass was triturated with isopropanol until free powder wasobtained, (11 g, yield 80%) λmax (water) 275 nm: ¹ H NMR (D₂ O) δ 8.13(s, 1 H, 4-H), 8.03 (dd. 1 H, J=9.0, 1.1 Hz, 6-H), 7.2 (d, 1 H, J=9.0Hz, 7-H), 4.51 (t, 2 H, J=7.5 Hz, α--CH₂), 2.25 (t, 2 H, J=7.5 Hz,γ--CH2₂), 1.99 (m, 2 H, β--CH2--), 1.35-1.66 (m, 4 H, δ--CH₂, γ--CH₂),1.61 (s, 6 H, --(CH₃)₂). R_(f) =0.55 (C-18, water-methanol, 25%).

3. Synthesis of Intermediate: A solution of1-(γ-carboxypentynyl)-2,3,3-trimethylindoleninium-5-sulfonate (IV) (10g, 0.03 mol) and N, N'diphenylformamidine (7.2 g, 0.04 mol) in aceticacid (20 mL) was heated to reflux for 1 h. Acetic acid was removed on arotary evaporator and the product washed with ethyl acetate (3×50 mL)whereupon a dark brown solid was obtained. λmax (water) 415 nm, R_(f)=0.32 (C18, 25% methanol in water). The crude product thus obtained wasused for the next reaction without further purification. The solid (3.8g) was dissolved in a mixture of acetic anhydride (10 mL) and, pyridine(5 mL). 5-Phthalimidomethyl-1-(ε-carboxypentynyl)-2,3,3-trimethylindole,(III)(2.6 g, 6 mmol) was added and the reaction mixture was heated at110° C. for 1 h. The solution was cooled and diluted with severalvolumes of diethyl ether (500 mL) . Product separated in the form of redpowder from which supernatant fluid was removed by decantation. It wasdissolved in a minimum volume of methanol and reprecipitated with2-propanol. The product was collected on a filter paper and dried toyield 5.3 g of compound (V). It was purified by flash columnchromatography on reverse-phase C18 using a water-methanol mixture aseluent, (1.6 g, yield 30%). λmax (water) 554 nm, ε 1.3×10⁵ L/mol-cm. ¹ HNMR (CD₃ OD) δ 8.5 (t, 1 H, J=14 Hz, β-proton of the bridge); 7.8-8.0(m, 6 H, 4 protons of phthalimido group & 4-H & 6-H of sulfoindolering), 7.55 (s, 2 H, 4'-H); 7.6 (d, 1 H, J=12 Hz, 6'-H); 7.3 (two d. 2H, 7-H & 7'-H); 6.1-6.3 (t, 2 H, α α' protons of the bridge); 4.1 (m, 4H. α & α' --CH₂ --); 2.9 (t, 2 H, J=7 Hz, --CH₂ COOH); 1.4-2.0 (m, 21 H,three --CH₂, one --CH₃ and two --(CH₃)₂), methyl protons of themethylphthalimido group are merged in a water signal at 4.8.

4. Hydrolysis of (V) to Give (VI). (1 g. 1.1 mmol) was dissolved inconcentrated hydrochloric acid (5 mL) and heated under reflux for 12 h.After cooling, the crystalline phthalic acid was filtered off. Thefiltrate was concentrated with a rotary evaporator and then slowlyneutralized with concentrated ammonium hydroxide while the temperaturewas kept below 30° C. Pure fluorochrome CY3NH₂ SO₃ (VI) was obtained byreverse phase (C18) column chromatography using a water-methanol mixtureas eluent. λmax (methanol) 552 nm, ¹ H NMR (DMSO, d₆) δ 8.45 (t, J=7.2Hz, 1 H, 9-H); 7.3-7.9 (m, 6 H, aromatic protons); 6.55 (dd, 2 H, 8 &8'-H); 4.5 (m, 4 H, N--CH₂); 4.1 (s, 2 H, CH₂ NH₂); 2.15 (t, 2 H, CH₂COOH); 1.25-1.8 (broad m, 24 H, 2--(CH₂)2 & 6--C--(CH₃)₂). R_(f) 0.415(RP C18 60% methanol in water)

5. Synthesis of complex 1. Dry powder of CY5(SO₃)₂ succinimidylester(425 mg, 0.26 mmol) was added in small portions to a well stirredsolution of CY3NH₂ SO₃ (200 mg, 0.26 mmoles) in 10 mL ofcarbonate-bicarbonate buffer (0.1 M, pH 9.4). Stirring was continued foradditional 30 minutes after which the reaction mixture was purified byflash column chromatography on C18 reverse phase powder usingwater:methanol (6.3:3.7) as solvent as eluent. 5 mL fractions werecollected and monitored by TLC. Fractions containing CY5(SO₃)₂ acid andCY3NH₂ SO₃ were discarded. Violet colored fractions were checked byultraviolet light in methanol and the fractions containing complex 1fluorochrome (FIG. 3) were pooled. Evaporation of the solvent yielded150 mg of complex 1 as violet powder (37%) R_(f) =0.45 (RP 37% methanolin water) fluorochrome 1:1 yield 37%. ¹ H NMR spectrum recorded in D₂ Oshowed broad signals and were difficult to assign. The fluorochrome waspurified on a strongly acidic ion-exchange column (Dowex 50) to removecationic counter ions. High resolution FAB mass spectrometry showed(M+H)⁺ ion at 1391.83 (C₇₃ H₉₁ N₅ O₁₆ S₃ +H requires 1391.73).

6. Succinimidyl Ester of Energy Transfer Cyanine Dye Complex 1 (60 mg,0.04 mmol) was dissolved in a mixture of dry DMF (1 mL), and drypyridine (0.05 mL). Disuccinimidyl carbonate (DSC) (46 mg, 0.18 mmol 1.5equiv/carboxyl group was added and the mixture was stirred at 55-60° C.for 90 min. under nitrogen. After diluting the mixture with dry diethylether (20 mL), the supernatant was decanted. The product was washedrepeatedly with ether, filtered and dried under vacuum. The formation ofthe active succinimidyl ester was confirmed by its reaction withbenzylamine in DMF or its reaction with taurine in a pH 9.4 bicarbonatebuffer. Reversed phase C18 TLC spotted with the conjugate, thesuccinimidyl ester and hydrolyzed carboxylate product for comparison wasdeveloped with water-methanol (1:1) mixture. R_(f) =0.78 (Acid), 0.3(Benzylamine adduct).

7. Reaction of Succinimidyl Ester with Antibody and Streptavidin. Astock solution of the complex 1 fluorochrome succinimidyl active esterwas made in dry DMF (1 mg/100 mL). In one sample, one milligram Sheepγ-globulin was dissolved in 0.25 mL carbonate/bicarbonate buffer(approximately 6.45 mmol/0.25 mL). In another, streptavidin wasdissolved in 0.25 mL of the carbonate/bicarbonate buffer. Appropriatevolumes of the fluorochrome stock were added to 0.25 mL portions of eachprotein solution to produce desired starting fluorochrome to antibodyratios, and each reaction mixture was stirred at room temperature for 30minutes. The protein conjugate was separated from unreacted fluorochromein each sample by gel filtration chromatography over sephadex G-50(0.7×20 cm column), using PBS, pH 7.4, containing 0.1% azide. Dyeconjugated proteins eluted as colored bands well separated from theunreacted fluorochrome. The normalized spectrum of the complex1-streptavidin conjugage in PBS is shown in FIG. 5. The absorbancespectrum of complex 1-Sheep IgG in PBS is shown in FIG. 6. FIG. 7 showsthe flow cytometry analysis of complex 1-streptavidin used to detect CD3antibody.

EXAMPLE 3

Several other complexes were synthesized.

FIGS. 8 and 9 shown the absorbance and emission spectra, respectively,for the complex fluorescein-CY3NH₂ SO₃ in methanol having the structure.##STR21## Excitation was at 488 nm with fluorescence emission at 574 nm.The quantum yield was 0.041, the Stokes shift was 74 and the %efficiency of the energy transfer was 98.3%. The absorbance max. foreach of the fluorochromes in the complex is 500 and 558. FIGS. 10 and 11shown the absorbance and emission spectra, respectively, for the complexfluorescein-CY3(NH₂)₂ -CY5(SO₃)₂ in methanol. The absorbance max. foreach of the fluorochromes is 500, 560 and 650. Excitation was at 488 nmand emission at 672 nm. The quantum yield was 0.1566, Stokes shift was172 and the % efficiency of the energy transfer was 99%. FIGS. 12 and 13shown the absorbance and emission spectra, respectively, for the complexfluorescein-CY3(NH₂)₂ -CY7(SO₃)₂. The absorbance max. for eachfluorochrome is 500, 560 and 754. Excitation was as 488 nm and emissionat 782 nm. The Stokes shift was 282 and the % efficiency was 99%. Theseseries of spectra demonstrate the efficient energy transfer with longStokes shifts. Each emission spectrum shows substantially all of theemission coming from the final acceptor fluorochrome in each series withonly minimal emission from either the donor fluorescein in FIG. 9 or theintermediate cyanine in FIGS. 11 and 13.

Multiparameter analyses can be done of multiple samples to detect thepresence of target biological compounds. Each sample is labeled by wellknown labeling methods with a different complex. For example, one samplesuspected of containing a target biological compound is incubated with asingle fluorochrome, such as fluorescein, cascade blue, a BODIPY dye orone of the monomethine rigidized dyes or CY3O(SO₃)₂ or CY3(SO₃)₂, allemitting in he 500-575 nm wavelength range (green to orange). A secondsample suspected of containing the target biological compound (the samecompound or a different compound as that in sample 1), is incubated witha complex of the invention, for example fluorescein-CY3NH₂ which willabsorb light at 488 nm and emits fluorescence at 574 nm (orange).Additional samples suspected of containing another target compound areincubated with other labeling complexes of the invention, such asfluorescein-CY3-CY5 and fluorescein-CY3-CY7 both of which absorb lightat 488 nm, but emit fluorescence at 672 nm and 782 nm, respectively (redto deep red). After a suitable period to permit the fluorescent labelsto bind with the target compounds, unbound label is washed and thelabeled samples are mixed. Detection is possible with a singlewavelength excitation source, i.e., at laser line 488 nm. Eachdifferently labeled sample will fluoresce a different color at theemission wavelength of its particular label. Those skilled in the artwill recognize that the fluorescent labeling complexes of the presentinventor can be used for a variety of immunofluorescent techniques,including direct and indirect immunoassays, or, competitive immunoassaysand other known fluorescence detection methods. The conditions of eachincubation, e.g., pH, temperature and time are known in the art, butgenerally room temperature is preferred. If reacting with a amine, pH9.4 is preferred. The pH is adjusted depending on the optimum reactionconditions for the particular reactive groups according to knowntechniques.

The fluorescent labeling complexes may be used to form reagents bycovalently binding the complexes to a carrier material, such as polymerparticles, cells, glass beads, antibodies, proteins, enzymes andnucleotides or nucleic acids (DNA and RNA) and analogs thereof whichhave been derivatized to include at least one first reactive groupcapable of forming a covalent bond with the functional group on thelabeling complex (or a functional group capable of forming a covalentbond with a reactive group on the complex, as described above) and atleast one second reactive group (or functional group, as the case maybe) having specificity for, and being capable of forming a covalent bondwith, a target biological compound, such as antibodies, cells, drugs,antigens, bacteria, viruses and other microorganisms. When the carrierhas functional groups, it may be antibody or DNA suited for attachmentto antigen or a complementary DNA sequence, respectively. When thecarrier material has reactive groups on it, the carrier may be a polymerparticle or an antigen suitable for attachment to DNA or an antibody,for example. Techniques for covalently binding fluorochromes to carriermolecules such as those mentioned are well known in the art and readilyavailable in the literature. The carrier material can further includenucleotide derivatized to contain one of an amino, sulfhydryl, carboxyl,carbonyl or hydroxyl groups, and oxy or deoxy polynucleic acidsderivatized to contain one of an amino, sulfhydryl, carboxyl, carbonylor hydroxyl groups. The functional groups on the carrier material whichare complementary to, i.e., form covalent bonds with, the reactivegroups of the labeling complexes of the invention include amino,sulfhydryl, carboxyl, hydroxyl and carbonyl groups.

A comparison of the energy transfer complex of the present invention tothe conventional R-Phycoerythrin dyes is shown in Table 6 below.

                  TABLE 6                                                         ______________________________________                                        COMPLEX 2 vs R-PHYCOERYTHRIN                                                               R-Phycoerythrin                                                                             Complex 2                                          ______________________________________                                        Excitation wavelength                                                                      488           488                                                  Emission wavelength 580 578                                                   488-laserline PE fluorescence signals were                                    Flow-Cytometer was greatly stable                                              reduced at pH 8.5 throughout pH                                               & extinguished range                                                          at pH 9.5                                                                    MW 240000 1667                                                                Staining do not penetrate labeled Ab                                           readily into penetrates                                                       intracellular into                                                            tissues to reach intracellular                                                target antigen tissues to reach                                                target antigen                                                              Binding Rate rate of binding rapid binding                                     to antigen is slow                                                         ______________________________________                                    

The energy transfer complexes of the present invention provide avaluable set of fluorescent labels which are particularly useful formultiparameter analysis and importantly, are sufficiently low inmolecular weight to permit materials labeled with the fluorescentcomplexes to penetrate all structures. As such, the complexes are wellsuited for use as DNA probes. The complexes of the invention and thereagents that can be made from them offer a wide variety of fluorescentlabels with large Stokes shifts. Those in the art will recognize thatthe complexes of the invention can be used in a variety of fluorescenceapplications over a wide range of the visible spectrum.

What we claim is:
 1. A low molecular weight fluorescent labeling complexfor labeling a target material consisting of:a first fluorochrome havingfirst absorption and emission spectra; a second fluorochrome havingsecond absorption and emission spectra, the wavelength of the emissionmaximum of said second fluorochrome being longer than the wavelength ofthe emission maximum of said first fluorochrome, and a portion of theabsorption spectrum of said second fluorochrome overlapping a portion ofthe emission spectrum of said first fluorochrome for transfer of energyabsorbed by said first fluorochrome when said complex is excited bylight to said second fluorochrome; a linker for covalently attachingsaid first and second fluorochromes capable of transfer of resonanceenergy between said first and second fluorochromes; at least one of saidfirst or second fluorochromes is a cyanine dye and the combinedmolecular weight of said first and second fluorochromes and said linkeris less than about 20,000 Daltons; and at least one group capable offorming a covalent bond with said target material.
 2. The complexrecited in claim 1 wherein said first fluorochrome has an extinctioncoefficient greater than 20,000 Liters per mole centimeter.
 3. Thecomplex recited in claim 1 wherein said second fluorochrome has afluorescence quantum yield greater than or equal to 0.05.
 4. A lowmolecular weight fluorescent labeling complex for labeling a targetmaterial consisting of:a first fluorochrome having first absorption andemission spectra; a second fluorochrome having second absorption andemission spectra, the wavelength of the emission maximum of said secondfluorochrome being longer than the wavelength of the emission maximum ofsaid first fluorochrome, and a portion of the absorption spectrum ofsaid second fluorochrome overlapping a portion of the emission spectrumof said first fluorochrome for transfer of energy absorbed by said firstfluorochrome to said second fluorochrome when said complex is excited bylight; a linker for covalently attaching said first and secondfluorochromes for transfer of resonance energy between said first andsecond fluorochromes; at least one of said first or second fluorochromesis a cyanine dye and the combined molecular weight of said first andsecond fluorochromes and said linker is less than about 20,000 Daltons;at least one group capable of forming a covalent bond with said targetmaterial; and, water solubilizing constituents, said water solubilizingconstituents being unreactive with said group capable of forming acovalent bond with said target material.
 5. The complex recited in claim4 wherein said solubilizing constituents are selected from the groupconsisting of amide, sulfonate, sulfate, phosphate, quaternary ammonium,hydroxyl and phosphonate.
 6. The complex recited in claim 1 wherein saidgroup is a reactive group that reacts with said target material and saidtarget material has functional groups selected from the group consistingof amino, hydroxyl, sulfhydryl, carboxyl and carbonyl.
 7. The complexrecited in claim 6 wherein said reactive group is selected from thegroup consisting of succinimidyl ester, isothiocyanate, isocyanate,iodoacetamide, dichlorotriazine, maleimide, sulfonyl halide,alkylimidoester, arylimidoester substituted hydrazines, substitutedhydroxylamines, carbodiimides, acid halide, and phosphoramidite.
 8. Thecomplex recited in claim 1 wherein the combined molecular weight of saidfirst and second fluorochromes and said linker is within the range ofabout 500 to about 10,000 Daltons.
 9. A low molecular weight fluorescentlabeling complex for labeling a target material consisting of:a firstfluorochrome having first absorption and emission spectra; a secondfluorochrome having second absorption and emission spectra, thewavelength of the emission maximum of said second fluorochrome beinglonger than the wavelength of the emission maximum of said firstfluorochrome, and a portion of the absorption spectrum of said secondfluorochrome overlapping a portion of the emission spectrum of saidfirst fluorochrome for transfer of energy absorbed by said firstfluorochrome when said complex is excited by light to said secondfluorochrome; a linker for covalently attaching said first and secondfluorochromes for transfer of resonance energy between said first andsecond fluorochromes; at least one of said first or second fluorochromesis a cyanine dye and the combined molecular weight of said first andsecond fluorochromes and said linker is less than about 20,000 Daltons;at least one group capable of forming a covalent bond with said targetmaterial; and, a third fluorochrome having third absorption and emissionspectra covalently attached through a linker to said secondfluorochrome; the wavelength of the emission maximum of said thirdfluorochrome being longer than the wavelength of the emission maximum ofsaid second fluorochrome, and a portion of the emission spectrum of saidsecond fluorochrome overlapping a portion of the absorption spectrum ofsaid third fluorochrome for transferring energy absorbed from said firstfluorochrome to said second fluorochrome to said third fluorochrome. 10.The complex recited in claim 9 wherein said first fluorochrome isselected from the group consisting of monomethine rigidized cyaninedyes, a trimethine cyanine dye, fluorescein, pyrene trisulfonate,bispyrromethene boron difluoride dyes and said second and thirdfluorochromes are polymethine cyanine dyes.
 11. A low molecular weightfluorescent labeling complex for labeling a target material consistingof:a plurality of first fluorochromes, each having first absorption andemission spectra; a second fluorochrome having second absorption andemission spectra, the wavelength of the emission maximum of said secondfluorochrome being longer than the wavelength of the emission maximum ofeach said first fluorochrome, and a portion of the absorption spectrumof said second fluorochrome overlapping a portion of the emissionspectrum of each said first fluorochrome for transfer of energy absorbedby each said first fluorochrome to said second fluorochrome when saidcomplex is excited by light; a plurality of linkers for covalentlyattaching said first fluorochromes to said second fluorochrome fortransfer of resonance energy from each said first fluorochromes to saidsecond fluorochrome upon excitation with light; at least one of saidfirst or second fluorochromes is a cyanine dye and the combinedmolecular weight of one of said first fluorochromes, said secondfluorochrome and said linker is less than about 20,000 Daltons; and, atleast one group capable of forming a covalent bond with said targetmaterial.
 12. A low molecular weight fluorescent labeling complex forlabeling a target material consisting of:a plurality of firstfluorochromes, each having first absorption and emission spectra; asecond fluorochrome having second absorption and emission spectra, thewavelength of the emission maximum of said second fluorochrome beinglonger than the wavelength of the emission maximum of each said firstfluorochrome, and a portion of the absorption spectrum of said secondfluorochrome overlapping a portion of the emission spectrum of each saidfirst fluorochrome for transfer of energy absorbed by each said firstfluorochrome to said second fluorochrome when said complex is excited bylight; a plurality of linkers for covalently attaching said firstfluorochromes to said second fluorochrome for transfer of resonanceenergy from each said first fluorochromes to said second fluorochromeupon excitation with light; at least one of said first or secondfluorochromes is a cyanine dye and the combined molecular weight of oneof said first fluorochromes, said second fluorochrome and said linker isless than about 20,000 Daltons; at least one group capable of forming acovalent bond with said target material; and, water solubilizingconstituents, said water solubilizing constituents being unreactive withsaid group capable of forming a covalent bond with said target material.13. The complex recited in claim 11 wherein said group capable offorming covalent bonds is a reactive group that reacts with said targetmaterial and said target material has functional groups selected fromthe group consisting of amino, hydroxyl, sulfhydryl, carbonyl andcarboxyl.
 14. The complex recited in claim 13 wherein said reactivegroup is selected from the group consisting of succinimidyl ester,isothiocyanate, isocyanate, iodoacetamide, dichlorotriazine, maleimide,sulfonyl halide, alkylimidoester, arylimidoester substituted hydrazines,substituted hydroxylamines, carbodiimides, acid halide, andphosphoramidite.
 15. A low molecular weight fluorescent labeling complexfor labeling a target material consisting of:a first fluorochrome havingfirst absorption and emission spectra; a plurality of secondfluorochromes each having second absorption and emission spectra, thewavelength of the emission maximum of each said second fluorochromebeing longer than the wavelength of the emission maximum of said firstfluorochrome, and a portion of the absorption spectrum of each saidsecond fluorochrome overlapping a portion of the emission spectrum ofsaid first fluorochrome and each being capable of accepting energy fromsaid first fluorochrome when said first fluorochrome is excited bylight; a plurality of linkers for covalently attaching said firstfluorochrome to each of said second fluorochromes for transfer ofresonance energy from said first fluorochrome to each of said secondfluorochromes; at least one of said first or second fluorochromes is acyanine dye and the combined molecular weight of said first and secondfluorochromes and said linker is less than about 20,000 Daltons; and atleast one group capable of forming a covalent bond with said targetmaterial.
 16. A low molecular weight fluorescent labeling complex forlabeling a target material consisting of:a first fluorochrome havingfirst absorption and emission spectra; a second fluorochrome havingsecond absorption and emission spectra, the wavelength of the emissionmaximum of said second fluorochrome being longer than the wavelength ofthe emission maximum of said first fluorochrome, and a portion of theabsorption spectrum of said second fluorochrome overlapping a portion ofthe emission spectrum of said first fluorochrome for transfer of energyabsorbed by said first fluorochrome when said complex is excited bylight to said second fluorochrome; a linker for covalently attachingsaid first and second fluorochromes for transfer of resonance energybetween said first and second fluorochromes; at least one of said firstor second fluorochromes is a cyanine dye and the combined molecularweight of said first and second fluorochromes and said linker is lessthan about 20,000 Daltons; at least one group capable of forming acovalent bond with said target material; and, water solubilizingconstituents, said water solubilizing constituents being unreactive withsaid group capable of forming a covalent bond with said target material.17. The complex recited in claim 15 wherein said group capable offorming covalent bonds is a reactive group that reacts with said targetmaterial and said target material has functional groups selected fromthe group consisting of amino, hydroxyl, sulfhydryl, carbonyl andcarboxyl.
 18. The complex recited in claim 17 wherein said reactivegroup is selected from the group consisting of succinimidyl ester,isothiocyanate, isocyanate, iodoacetamide, dichlorotriazine, maleimide,sulfonyl halide, alkylimidoester, arylimidoester, substitutedhydrazines, substituted hydroxylamines, carbodiimides, acid halide, andphosphoramidite.
 19. The complex recited in claim 1 wherein said linkeris a linker of about 2 to 20 bond lengths.
 20. The complex recited inclaim 1 wherein said group capable of forming covalent bonds is afunctional group capable of covalently binding to a reactive group onthe target material, said functional group being selected from the groupconsisting of amino, sulfhydryl, carbonyl, carboxyl and hydroxy groupsand said reactive group being selected from the group consisting ofsuccinimidyl ester, isothiocyanate, isocyanate, iodoacetamide,dichlorotriazine, maleimide, sulfonyl halide, alkylimidoester,arylimidoester substituted hydrazines, substituted hydroxylamines,carbodiimides, acid halide, and phosphoramidite.